IL-12 is required for immunity to Leishmaniasis. We have previously reported that the infection of mouse bone-marrow- derived or inflammatory macrophages with Leishmania leads to a selective impairment of IL-12-inducing pathways. We have argued that as a consequence, infected macrophages will drive early T cell activation in the absence of the major physiologic inducer of IFN-g, and that this effect explains the delayed onset of cell-mediated control mechanisms that is typical of even self- limiting forms of leishmaniasis. We have extended these studies to include an analysis of cytokine production by infected epidermal Langerhans cells (LC), since it is these cells that have been implicated in the initiation of immune responses in the skin. Using a culture system that allows Langerhans cell-like cells to be routinely expanded and isolated from C57BL/6 fetal skin (fetal skin-derived dendritic cells-FSDDC) we have begun to study dendritic cell-parasite interactions. L. major amastigotes, but not metacyclic promastigotes, were readily internalized by FSDDC. Parasite internalization was associated with activation of FSDDC manifested by upregulation of MHC class I and II surface antigens, increased expression of co-stimulatory molecules (CD40, CD54, and CD86) and IL-12 release all over an 18-hr time period. Although tissue macrophages also readily internalized amastigotes, parasite internalization did not lead to similar changes in surface antigen expression or cytokine production. The data suggest that activation of, and IL-12 induction in, skin dendritic cells by L. major amastigotes is important for development of protective Th1 predominant anti- Leishmania immunity, and that so long as infections are confined to macrophages, the onset of cell-mediated immune responses will be avoided. Studies have been completed that evaluated the toxicity, immunogenicity and efficacy of a clinical grade killed Leishmania vaccine made by Biobras in Brazil plus rhuIL-12 in monkeys (Macaca mulatta). Twelve monkeys receiving two intradermal inoculations of alum absorbed killed vaccine plus IL-12 were completely protected against homologous challenge with L. amazonensis. All but one of 16 animals in the control groups developed typical cutaneous ulcers. The protection was correlated with high levels of IFN-g in serum and in culture supernatants of PBLs stimulated with antigen in vitro. Vaccination with killed antigen using IL-12 and alum as adjuvants was safe and effective in this primate model. Phase I clinical trials are planned. Despite the dramatic increase in the incidence of unresponsiveness to antimony therapy in Indian kala-azar, the possibility that this might be due to the emergence of antimony resistant strains of L. donovani has not been demonstrated. The susceptibility of L. donovani isolates to sodium antimony gluconate (SAG) was compared using infected macrophages in vitro. Isolates were obtained from splenic biopsies of patients in Bihar, India, who either did not respond (18) or did respond (10) to one or more full courses of treatment with SAG. A strong correlation (p < 0.005) with clinical response was observed only when strains were assayed as intracellular amastigotes; responsive isolates ED/50\= 2.5 ?2.8 ?g/ml; unresponsive isolates ED/50\ = 7.2?4 ug/ml. From these data we conclude that treatment failures in India Kala-azar are due to infection with antimony resistant strains of L. donovani. Between July 1, 1997, and June 30, 1998, five patients with suspected leishmaniasis were seen and evaluated at the NIH clinical center. Four of the five were cases of cutaneous disease; one was a Peace Corps volunteer from Mauritania, one was an ornithologist working in the Manu Forest in Peru, one was an elderly woman from Pakistan, and the fourth cutaneous case was a tourist whose lesions healed spontaneously. The most interesting patient was a native Zairean with visceral leishmaniasis, probably acquired in the Aegean Islands of Greece when the patient was attending Athens University. One new feature of the leishmaniasis experience of the past year was the use of liposome-encapsulated amphotericin (Ambisome) for treatment of two of the cutaneous infections as well as the visceral case. The advantage of AmBisome treatment is the shorter duration of treatment (5 or 6 doses over 10 days) as compared to Pentostam (18-20 days).